Single-cell mRNA sequencing of giant panda (Ailuropoda melanoleuca) seminoma reveals the cellular and molecular characteristics of tumour cells.

Testicular tumours are zoonoses that can occur in not only human, but other animals, include giant pandas. A middle-aged male giant panda named Fufu was diagnosed with a testicular tumour and underwent surgery to remove the entire left testis. The testis was mainly composed of three substantive parts: normal tissue on the outside, tumour tissue in the middle, and necrosis in the centre. HE stains revealed that the tumour was a seminoma. Single-cell mRNA sequence was applied to characterise cellular states and molecular circuitries of giant panda testicular seminoma. Only germ cell markers expressed in nearly all tumour cells, and the tumour cells appeared to be the same subtype of seminoma cells. We identified four clusters with unique genes expression. They were early apoptosis cells (EAC), inactive cells (IC), active cells subcluster 1 (AC-1) and active cells subcluster 2 (AC-2). We utilised monocle tools and found that IC cells was in the initiation stage, and EAC was one type of terminal stage, suggesting that tumour cells may undergo apoptosis in the future. AC-2 was another type of terminal stage, representing a group of progressive cells. Our study represents the first report to utilise scRNA-seq to characterise the cellular states and molecular circuitries of a giant panda testicular tumour. This investigation proposes CD117 and CD30 as dependable markers for future pathologic diagnosis. Our findings also suggest that CTSV and other genes with unique expression patterns in active and progressive giant panda seminoma cells may act as early prognostic biomarkers.

The bear necessities: A sensitive qPCR assay for bear DNA detection from bile and derived products to complement wildlife forensic enforcement.

Demand for bear bile, a prized component of traditional Asian medicines, threaten Asiatic and sun bear population sustainability. While laws exist to prevent poaching and trafficking of bear parts and derivatives, smuggling persists with demand extending to surrogate species, including American black bears (Ursus americanus). Mitochondrial DNA (mtDNA) sequencing can identify products putatively containing biological bear material but can be undermined by PCR inhibitors in bile and a lack of sensitivity at trace levels. Quantitative PCR (qPCR) assays can be used to distinguish between closely related target species, while concomitantly evaluating inhibition and false negative results in low quality/quantity DNA applications. Herein, we develop a multiplexed qPCR assay to detect and differentiate among bear species, including highly diluted bile samples mixed within liquors as common dilutants. The assay detects as little as 10 locus copies/reaction of bear DNA with 95% confidence, distinguishing among sun, Asiatic and American black bears. Demonstrating the sensitivity and applicability of this assay in context of current bile mixture recipes, dilutions of 1:5,000 bile with ethanol, red wine, and spirits, all yielded clear quantifiable detections, where our data suggests as little as 1 drop of bile per 750 mL bottle of alcohol would still exceed the limits of detection (e.g., 1:15000 dilution or <0.05 mL bile per 750 mL bottle). Overall, this study provides a rapid, sensitive, and specific test to identify and distinguish among bear species commonly used for bile production to aid wildlife enforcement applications.

Circadian gene transcription plays a role in cellular metabolism in hibernating brown bears, Ursus arctos.

Hibernation is a highly seasonal physiological adaptation that allows brown bears (Ursus arctos) to survive extended periods of low food availability. Similarly, daily or circadian rhythms conserve energy by coordinating body processes to optimally match the environmental light/dark cycle. Brown bears express circadian rhythms in vivo and their cells do in vitro throughout the year, suggesting that these rhythms may play important roles during periods of negative energy balance. Here, we use time-series analysis of RNA sequencing data and timed measurements of ATP production in adipose-derived fibroblasts from active and hibernation seasons under two temperature conditions to confirm that rhythmicity was present. Culture temperature matching that of hibernation body temperature (34 °C) resulted in a delay of daily peak ATP production in comparison with active season body temperatures (37 °C). The timing of peaks of mitochondrial gene transcription was altered as were the amplitudes of transcripts coding for enzymes of the electron transport chain. Additionally, we observed changes in mean expression and timing of key metabolic genes such as SIRT1 and AMPK which are linked to the circadian system and energy balance. The amplitudes of several circadian gene transcripts were also reduced. These results reveal a link between energy conservation and a functioning circadian system in hibernation.

Characteristics of mesenchymal stem cells and their exosomes derived from giant panda (Ailuropoda melanoleuca) endometrium.

Conservation of genetic resources is an important way to protect endangered species. At present, mesenchymal stem cells (MSCs) have been isolated from the bone marrow and umbilical cords of giant pandas. However, the types and quantities of preserved cell resources were rare and limited, and none of MSCs was derived from female reproductive organs. Here, we first isolated MSCs from the endometrium of giant panda. These cells showed fibroblast morphology and expressed Sox2, Klf4, Thy1, CD73, CD105, CD44, CD49f, and CD105. Endometrium mesenchymal stem cells (eMSCs) of giant panda could induce differentiation into three germ layers in vitro. RNA-seq analysis showed that 833 genes were upregulated and 716 genes were downregulated in eMSCs compared with skin fibroblast cells. The results of GO and the KEGG analysis of differentially expressed genes (DEGs) were mainly focused on transporter activity, signal transducer activity, pathways regulating pluripotency of stem cells, MAPK signaling pathway, and PI3K-Akt signaling pathway. The genes PLCG2, FRK, JAK3, LYN, PIK3CB, JAK2, CBLB, and MET were identified as hub genes by PPI network analysis. In addition, the exosomes of eMSCs were also isolated and identified. The average diameter of exosomes was 74.26 ± 13.75 nm and highly expressed TSG101 and CD9 but did not express CALNEXIN. A total of 277 miRNAs were detected in the exosomes; the highest expression of miRNA was the has-miR-21-5p. A total of 14461 target genes of the whole miRNAs were predicted and proceeded with functional analysis. In conclusion, we successfully isolated and characterized the giant panda eMSCs and their exosomes, and analyzed their functions through bioinformatics techniques. It not only enriched the conservation types of giant panda cell resources and promoted the protection of genetic diversity, but also laid a foundation for the application of eMSCs and exosomes in the disease treatment of giant pandas.

Microsporum gypseum Isolated from Ailuropoda melanoleuca Provokes Inflammation and Triggers Th17 Adaptive Immunity Response.

Microsporum gypseum causes dermatomycoses in giant pandas (Ailuropoda melanoleuca). This study aimed to investigate the immune response of M. gypseum following deep infection. The degree of damage to the heart, liver, spleen, lungs, and kidneys was evaluated using tissue fungal load, organ index, and histopathological methods. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detected the mRNA expression of receptors and cytokines in the lung, and immunofluorescence staining and flow cytometry, were used to assess immune cells in the lung. The results indicated that conidia mainly colonized the lungs and caused serious injury with M. gypseum infection. Furthermore, dectin-1, TLR-2, and TLR-4 played a role in recognizing M. gypseum cells. Numerous inflammatory cells, mainly macrophages, dendritic cells, polymorphonuclear neutrophils, and inflammatory cytokines (TGF-ß, TNF-α, IL-1ß, IL-6, IL-10, IL-12, and IL-23), were activated in the early stages of infection. With the high expression of IL-22, IL-17A, and IL-17F, the Th17 pathway exerted an adaptive immune response to M. gypseum infection. These results can potentially aid in the diagnosis and treatment of diseases caused by M. gypseum in giant pandas.

Comparison of transcriptome profiles of mesenchymal stem cells derived from umbilical cord and bone marrow of giant panda (Ailuropoda melanoleuca).

Mesenchymal stem cells (MSCs) have pluripotent differentiation ability and play an important role in human clinical cell therapy. While, the research on MSCs in endangered wild animals is extremely rare. In our previous studies, the bone marrow mesenchymal stem cells (bmMSCs) and umbilical cord mesenchymal stem cells (ucMSCs) of giant panda (Ailuropoda melanoleuca) were successfully isolated. We aimed to characterize the differences in gene expression profiles between these two types of MSCs using RNA sequencing (RNA-Seq) and to determine which potential pathways are involved in functional regulation. In total, 1079 significantly differentially expressed genes (DEGs) were identified, of which 478 genes were upregulated and 601 genes were downregulated. The significantly enriched Gene Ontology (GO) terms mainly contained cell adhesion, biological adhesion, intracellular signal transduction, molecular function regulator, Ras protein signal transduction, small GTPase mediated signal transduction, and regulation of Rho protein signal transduction. The most enrichment pathways of DEGs enriched in Kyoto Encyclopedia of Genes Genomes (KEGG) were PI3K-AKT signaling pathway, Rap1 signaling pathway, MAPK signaling pathway, Hippo signaling pathway, Wnt signaling pathway, cGMP-PKG signaling pathway and Signaling pathways regulating pluripotency of stem cells. In addition, quantitative real time polymerase chain reaction (qRT-PCR) showed that the AKT3, CDK2, MAPK3, mTOR, PI3K and PTK2 genes associated with PI3K-AKT pathway were highly expressed (P < 0.01), and Caspase-3 was low expressed (P < 0.05) in ucMSCs group when compared with bmMSCs. After treatment with the PI3K inhibitor LY294002, genes AKT3, CDK2, MAPK3, mTOR, and PTK2 were significantly decreased in ucMSCs (P < 0.01), and Caspase-3 was significantly up regulated (P < 0.001). In conclusion, we for the first time compared and analyzed the transcriptome profiles of giant panda ucMSCs and bmMSCs, and found the PI3K-AKT pathway was highly activated and might be a key signaling pathway in the ucMSCs regulation. This study will be beneficial for the research on MSCs proliferation regulation and differentiation of giant pandas in the future, and lay the foundation for MSCs application and clinical therapy for endangered wild animals.

Temporal Analysis of Gene Expression and Isoform Switching in Brown Bears (Ursus arctos).

Hibernation in brown bears is an annual process involving multiple physiologically distinct seasons-hibernation, active, and hyperphagia. While recent studies have characterized broad patterns of differential gene regulation and isoform usage between hibernation and active seasons, patterns of gene and isoform expression during hyperphagia remain relatively poorly understood. The hyperphagia stage occurs between active and hibernation seasons and involves the accumulation of large fat reserves in preparation for hibernation. Here, we use time-series analyses of gene expression and isoform usage to interrogate transcriptomic regulation associated with all three seasons. We identify a large number of genes with significant differential isoform usage (DIU) across seasons and show that these patterns of isoform usage are largely tissue-specific. We also show that DIU and differential gene-level expression responses are generally non-overlapping, with only a small subset of multi-isoform genes showing evidence of both gene-level expression changes and changes in isoform usage across seasons. Additionally, we investigate nuanced regulation of candidate genes involved in the insulin signaling pathway and find evidence of hyperphagia-specific gene expression and isoform regulation that may enhance fat accumulation during hyperphagia. Our findings highlight the value of using temporal analyses of both gene- and isoform-level gene expression when interrogating complex physiological phenotypes and provide new insight into the mechanisms underlying seasonal changes in bear physiology.

A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears.

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

Using Gene Transcription to Assess Ecological and Anthropological Stressors in Brown Bears.

Increasingly, population- and ecosystem-level health assessments are performed using sophisticated molecular tools. Advances in molecular technology enable the identification of synergistic effects of multiple stressors on the individual physiology of different species. Brown bears (Ursus arctos) are an apex predator; thus, they are ideal candidates for detecting potentially ecosystem-level systemic perturbations using molecular-based tools. We used gene transcription to analyze 130 brown bear samples from three National Parks and Preserves in Alaska. Although the populations we studied are apparently stable in abundance and exist within protected and intact environments, differences in transcript profiles were noted. The most prevalent differences were among locations. The transcript patterns among groups reflect the influence of environmental factors, such as nutritional status, disease, and xenobiotic exposure. However, these profiles also likely represent baselines for each unique environment by which future measures can be made to identify early indication of population-level changes due to, for example, increasing Arctic temperatures. Some of those environmental changes are predicted to be potentially positive for brown bears, but other effects such as the manifestation of disease or indirect effects of oceanic acidification may produce negative impacts.

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

Transcriptome-Derived Tetranucleotide Microsatellites and Their Associated Genes from the Giant Panda (Ailuropoda melanoleuca).

Recently, an increasing number of microsatellites or simple sequence repeats (SSRs) have been found and characterized from transcriptomes. Such SSRs can be employed as putative functional markers to easily tag corresponding genes, which play an important role in biomedical studies and genetic analysis. However, the transcriptome-derived SSRs for giant panda (Ailuropoda melanoleuca) are not yet available. In this work, we identified and characterized 20 tetranucleotide microsatellite loci from a transcript database generated from the blood of giant panda. Furthermore, we assigned their predicted transcriptome locations: 16 loci were assigned to untranslated regions (UTRs) and 4 loci were assigned to coding regions (CDSs). Gene identities of 14 transcripts contained corresponding microsatellites were determined, which provide useful information to study the potential contribution of SSRs to gene regulation in giant panda. The polymorphic information content (PIC) values ranged from 0.293 to 0.789 with an average of 0.603 for the 16 UTRs-derived SSRs. Interestingly, 4 CDS-derived microsatellites developed in our study were also polymorphic, and the instability of these 4 CDS-derived SSRs was further validated by re-genotyping and sequencing. The genes containing these 4 CDS-derived SSRs were embedded with various types of repeat motifs. The interaction of all the length-changing SSRs might provide a way against coding region frameshift caused by microsatellite instability. We hope these newly gene-associated biomarkers will pave the way for genetic and biomedical studies for giant panda in the future. In sum, this set of transcriptome-derived markers complements the genetic resources available for giant panda.

Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears.

Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period.

Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.

Comparative analysis and molecular characterization of genomic sequences and proteins of FABP4 and FABP5 from the giant panda (Ailuropoda melanoleuca).

Fatty acid binding proteins (FABPs) are a family of small, highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. In this study, cDNA and genomic sequences of FABP4 and FABP5 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using reverse transcription polymerase chain reaction (RT-PCR) technology and touchdown-PCR. The cDNAs of FABP4 and FABP5 cloned from the giant panda were 400 and 413 bp in length, containing an open reading frame of 399 and 408 bp, encoding 132 and 135 amino acids, respectively. The genomic sequences of FABP4 and FABP5 were 3976 and 3962 bp, respectively, which each contained four exons and three introns. Sequence alignment indicated a high degree of homology with reported FABP sequences of other mammals at both the amino acid and DNA levels. Topology prediction revealed seven protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, two N-myristoylation sites, and one cytosolic fatty acid-binding protein signature in the FABP4 protein, and three N-glycosylation sites, three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one N-myristoylation site, one amidation site, and one cytosolic fatty acid-binding protein signature in the FABP5 protein. The FABP4 and FABP5 genes were overexpressed in Escherichia coli BL21 and they produced the expected 16.8- and 17.0-kDa polypeptides. The results obtained in this study provide information for further in-depth research of this system, which has great value of both theoretical and practical significance.

Overexpression, purification, molecular characterization and the effect on tumor growth of ribosomal protein L22 from the Giant Panda (Ailuropoda melanoleuca).

The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 µg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 µg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 µg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.

Polar bears exhibit genome-wide signatures of bioenergetic adaptation to life in the arctic environment.

Polar bears (Ursus maritimus) face extremely cold temperatures and periods of fasting, which might result in more severe energetic challenges than those experienced by their sister species, the brown bear (U. arctos). We have examined the mitochondrial and nuclear genomes of polar and brown bears to investigate whether polar bears demonstrate lineage-specific signals of molecular adaptation in genes associated with cellular respiration/energy production. We observed increased evolutionary rates in the mitochondrial cytochrome c oxidase I gene in polar but not brown bears. An amino acid substitution occurred near the interaction site with a nuclear-encoded subunit of the cytochrome c oxidase complex and was predicted to lead to a functional change, although the significance of this remains unclear. The nuclear genomes of brown and polar bears demonstrate different adaptations related to cellular respiration. Analyses of the genomes of brown bears exhibited substitutions that may alter the function of proteins that regulate glucose uptake, which could be beneficial when feeding on carbohydrate-dominated diets during hyperphagia, followed by fasting during hibernation. In polar bears, genes demonstrating signatures of functional divergence and those potentially under positive selection were enriched in functions related to production of nitric oxide (NO), which can regulate energy production in several different ways. This suggests that polar bears may be able to fine-tune intracellular levels of NO as an adaptive response to control trade-offs between energy production in the form of adenosine triphosphate versus generation of heat (thermogenesis).

Overexpression, purification, and pharmacologic evaluation of anticancer activity of ribosomal protein L24 from the giant panda (Ailuropoda melanoleuca).

The ribosomal protein L24 (RPL24) belongs to the L24E family of ribosomal proteins and is located in the cytoplasm. The purpose of this study was to investigate the structure and anti-cancer function of RPL24 of the giant panda (Ailuropoda melanoleuca). The complementary DNA of RPL24 was cloned successfully using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing RPL24 complementary DNA and overexpressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified using Ni-chelating affinity chromatography. The results indicated that the length of the fragment cloned is 509 bp, and it contains an open-reading frame of 474 bp encoding 157 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL24 protein is 17.78 kDa with a theoretical isoelectric point of 11.86. The RPL24 gene is readily expressed in E. coli, and the RPL24 fused with the N-terminal histidine-tagged protein to give rise to the accumulation of an expected 23.51-kDa polypeptide. The inhibitory rate in mice treated with 0.1 mg/mL RPL24, the highest of 3 doses administered, can reach 67.662%, which may be comparable to the response to mannatide. The histology of organs with tumors showed that the tissues in the RPL24 group displayed a looser arrangement compared with that in the control group. Furthermore, no obvious damage was apparent in other organs, such as heart, lung, and kidney. The data showed that the recombinant RPL24 had time and dose dependency on the cell growth inhibition rate. Human laryngeal carcinoma Hep-2 cells treated with 0.3125-10 µg/mL RPL24 for 24 h displayed significant cell growth inhibition (P < 0.05; N = 6) in assays using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide compared with that in control (untreated) cells. By contrast, human hepatoma Hep G-2 cells displayed no significant change (P > 0.05; N = 6) from control (untreated) cells. RPL24 has time and dose dependency on Hep-2 cell growth inhibition. The data indicate that the effect at low concentrations is better than that at high concentrations, and the concentration of 0.625 µg/mL provides the best rate of growth inhibition. Further research is ongoing to determine the bioactive principles of recombinant RPL24 protein that are responsible for its anticancer activity.

Polymorphism of follicle stimulating hormone beta (FSHß) subunit gene and its association with litter traits in giant panda.

The different SSCP patterns of the follicle stimulating hormone beta (FSHß) gene amplified by three pairs of primers were sequenced. Comparisons among the three nucleotide sequences of three genotypes indicated that three base substitutions (A213T, A91G, and A89C) were detected in FSHß gene, which A213T substitution led to one amino acids mutation (Lys > Met), and the other two substitutions were synonymous mutations. The AA, AB and BB genotypes patterns obtained by FSHß primer1 had evident relation with the litter traits, but the SSCP genotypes patterns obtained by FSHß primer2 and primer3 had no evident relation with the litter traits in giant panda. The giant panda with AA and AB genotype had the largest litter size and multiparity rate compared with the BB genotypes (P < 0.05). We speculated that the giant pandas with the A allele have better litter traits than those with the B allele.

Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein.

To construct a fusion cytokine protein with more and stronger bioactivities to enhance the immunity of the cytokine alone, we expressed interleukin (IL)-6/(IL)-2 from giant panda (Ailuropoda melanoleuca) in Escherichia coli as a 59.4-kDa fusion protein. Subsequently, the inclusion bodies were solubilized with 8 M urea and applied onto a Ni-nitrilotriacetic acid column. The final production of IL-6/IL-2 reached 6 mg/L in soluble form, and the purified final product was >96% pure. In Western blot assays, the recombinant IL-6/IL-2 was recognized by polyclonal antibodies against IL-6 and IL-2 of giant panda. The results demonstrated that the protein mixture contained correctly folded IL-2 and IL-6 proteins. A 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay demonstrated that IL-6/IL-2 can promote lymphocyte proliferation and differentiation. These data suggest that the fusion protein could be used to develop a novel immunoadjuvant to enhance the immunity of animals against infectious diseases.

Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda (Ailuropoda Melanoleuca).

The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance.